Considerations for RT-qPCR in molecular point of care testing devices

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Time to read:   3 minute read

Updated : Fri, April 15, 2022 @ 8:58 PM

Originally published : Tue, Mar 01, 2022 @ 01:11 PM

The COVID-19 pandemic has accelerated the uptake of point of care testing (POCT), with simple, user-administered tests becoming commonplace in schools, places of work and people’s homes. Antigen POCT has been pivotal in SARS-CoV-2 detection, particularly where reverse transcription PCR (RT-PCR) cannot be delivered in a timely manner. However, the use of antigen tests isn’t always appropriate due to variable sensitivity.1 RT-PCRor RT-qPCR if using real-time detectionoffers greater sensitivity and specificity. Leveraging this technology in POCT brings lab quality results to the point of need, providing rapid, accurate and actionable results to clinicians. 

The rapid expansion in the use of POCT has fuelled the development of molecular point of care devices. Diagnostic manufacturers have seized this opportunity, by expanding assay development beyond SARS-CoV-2 detection, with panels for respiratory infections, sexually transmitted infections and antimicrobial resistance. 

The challenge for those looking to incorporate quality molecular diagnostics in a POCT device is ensuring accurate, reliable RT-PCR performance out of the stable, predictable conditions of a lab. 

Overcoming manufacturing challenges  

The manufacture of molecular point of care devices creates new demands for reagent and component suppliers. For example, glycerol-free reagents and hardy (typically thermostable) enzymes that can survive demanding lyophilisation processes and subsequent long term, ambient storage in various devices. 

To solve these manufacturing challenges, selecting an RT-qPCR master mix with the ideal properties for POCT devices is crucial. 

Here are some features and benefits to look out for: 

A one-step protocol 

RT-qPCR-1-step

The simplicity of a one-tube, one-step RT-qPCR master mix benefits POCT device manufacturers by eliminating an in-house pipetting step, which is typically the addition of an unstable reverse transcriptase to the final mix just prior to use. The removal of this step reduces the chance of contamination, errors and batch to batch variability. Crucially, in-house QC testing on the pre-combined master mix is significantly reduced since this is often handled by the component supplier. 

Concentrated for higher test sensitivity 

A 5X concentrated master mix creates valuable space saving benefits for miniaturised devices. Whether lyophilising within the device or manufacturing a separate bead, a 5X master mix offers greater flexibility for adding lyophilisation excipients. Reduced master mix volumes lead to smaller lyophilised pellets or beads, thereby allowing smaller channels to be used within devices.  

Thermostable and heat-activated 

Using a thermostable reverse-transcriptase that can withstand high template denaturation temperatures prior to cDNA synthesis may enable detection of challenging templates. For example double-stranded RNA viruses or those with a strong secondary structure. Heat-activated enzymes allow for processing at room temperature while preventing non-specific background amplification. 

Multiplex for efficiency 

While isothermal amplification methods are available for POCT devices, the options for multiplexing are limited and complex. 

RT-qPCR can target multiple regions of the same pathogen genome for higher sensitivity or detect a panel of pathogens with a multiplexed assay. This type of assay offers a more efficient use of resources, including patient samples, and a greater chance of achieving a comprehensive, specific diagnosis. For example, a multiplex test for influenza, RSV and SARS-CoV-2 for rapid diagnosis of patients with flu-like symptoms. 

Test and diagnose in one visit 

Point of care testing enables clinicians to choose and carry out an appropriate test, receive results and initiate treatment all in one visit with a patient. To meet this goal, the test must be fast. 

Advances in microfluidics and lab-on-a-chip technology have enabled the use of rapid PCR in POCT devices. The type, material and size of the reaction vessel and the method for thermal cycling are key parameters used to optimise the trade-off between speed and sensitivity.2 These factors, along with a stable, flexible and lyophilisation-compatible master mix are critical for manufacturing devices that deliver fast and accurate results. 

Partner with the right supplier 

Throughout the COVID-19 pandemic, LGC, Biosearch Technologies has served the diagnostic community as a reliable mission-critical component supplier for oligos, enzymes and sample preparation solutions.  

Our RapiDxFire Lyo-Flex 1-Step RT-qPCR 5x Master Mix has been created with input from top molecular diagnostic assay manufacturers to support assays requiring fast and accurate results. The master mix is designed to meet the challenges of bringing PCR based pathogen detection out of the lab and achieve predictable performance in unpredictable conditions. 

Whether you’re in the early stages of research or ready for commercialisation, partner with us as you develop, optimise and evolve your molecular diagnostics to meet current and future infectious disease detection needs. 

Learn more about RapiDxFire Lyo-Flex 1-Step RT-qPCR 5x Master Mix.

Learn more

References 

  1. Dinnes J, Deeks JJ, Berhane S, Taylor M, Adriano A, Davenport C, Dittrich S, Emperador D, Takwoingi Y, Cunningham J, et al. Rapid, point‐of‐care antigen and molecular‐based tests for diagnosis of SARS‐CoV‐2 infection. Cochrane Database of Systematic Reviews. John Wiley & Sons, Ltd. https://doi.org/10.1002/14651858.CD013705.pub2. Published 2021, Accessed 22 February 2022.
  2. Dong X, Liu L, Tu Y, Zhang J, Miao G, Zhang L, Ge S, Xia N, Yu D, Qiu X. Rapid PCR powered by microfluidics: A quick review under the background of COVID-19 pandemic. TrAC Trends in Analytical Chemistry. https://doi.org/10.1016/j.trac.2021.116377. Published 2021. Accessed 28 January 2022.

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